Pin M III gene in white pine

ABSTRACT

A gene coding for  Pin m  III, which is associated with overwintering and frost hardiness in western white pine, has been cloned and characterized. The gene contains an open reading frame of 486 base pairs, encoding a protein of 161 amino acid residues, with a calculated molecular weight of 18002.30 daltons. The predicted isoelectricl point is 5.534. The gene is transcriptionally regulated with the highest expression in fall and winter months. The protein  Pin m  III is high in infected tissue such as canker. Accordingly, the gene can be used to determine frost hardiness and blister resist fungus susceptibility in conifers.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a gene coding for Pin m III which is associated with overwintering and frost hardiness of western white pine.

The invention also relates to a method of determining the frost hardiness of a conifer seedling, and to a method for determining the susceptibility of conifers to blister rust fungus.

2. Description of the Prior Art

The following disclosure refers to a plurality of literature references. The references are listed numerically at the end of this disclosure

White pine blister rust is a disease of five-needle pines, e.g. western white pine (Pinus monticola D. Don), eastern white pine (Pinus strobes L.) and sugar pine (Pinus lambertiana Dougl.), caused by the blister rust fungus, Cronartium ribicola Fisch. The fungus has five different spore stages on two unrelated hosts, white pines and Ribes species. The basidiospores are produced on Ribes in the fall and transported by wind to pine foliage, where they germinate and infect the needle via stomata and produce infection spot. Hyphae grow down the needle into the bark, causing a perennial canker which eventually kills the tree. Although some large and older trees can survive, infected younger trees almost inevitably die. We have been studying proteins involved in this host-pathogen interaction. (e.g. Ekramoddoullah and Hunt, 1993, Ref. 10). During this investigation it was observed that environmental factors also contributed to the changes in the synthesis of proteins. Plants presumably respond to signals generated by environmental stresses, e.g. low and high temperature, photoperiod, drought, salinity and are capable of responding to a varying degree to the stresses (Bohnert et al., 1995, Ref. 3). Plants also are continually exposed to numerous microorganisms but are susceptible only to a few of them. In response to both abiotic and biotic stresses, induction of several proteins in plants have been identified (Heikila et al., 1984, Ref. 16; Ort et al., 1989, Ref. 32; Hightower, 1991, Ref. 17; Stintzi et al., 1993, Ref. 41; Ekramoddoullah et al., 1995, Ref. 12; Sabehat et al., 1996, Ref. 37; Mauch et al. 1988, Ref. 28). Although functions for many of these proteins have not yet been assigned, accumulation of a group of proteins termed “pathogenesis-related” proteins (van Loon, 1985, Ref. 48; Linthorst, 1991, Ref. 25) is considered an important feature of plant defence response upon infection. Some of these proteins could be induced by either abiotic or biotic stress. For example, a protein osmotin that accumulates during adaptation of tobacco cells to osmotic stress could be induced in tobacco leaves infected with tobacco mosaic virus (Stintzi et al., 1991, Ref. 40). Genes encoding vegetative storage proteins were expressed following wounding and water deficit (Mason et al., 1991, Ref. 27). A major protein, sporamin of potato tuberous roots (Maeshima et al., 1985, Ref. 26), which is developmentally regulated, could be induced in petioles by treatment with fungal elicitors, polygalacturonic acid and chitosan (Ohto et al., 1992, Ref. 31). A family of wound induced genes in Populus shares common features with genes encoding vegetative storage proteins (Davis et al., 1993, Ref. 7) that also accumulate seasonally in poplar bark tissues (Clausen and Apel, 1991, Ref. 5)). A group of antifreeze like proteins in rye grass was shown to have amino acid sequence homology with pathogenesis-related proteins (Hon et al., 1995, Ref. 18). Recently, a sugar pine protein Pin l I was detected in the foliage in increasing amounts in the fall (Ekramoddoullah et al. 1995, Ref. 12). The homologue of this fall protein, named as Pin m III, was also identified in western white pine foliage. An 89% homology of the N-terminal amino acid sequence of Pin m III was found with Pin l I (Ekramoddoullah and Taylor, 1996, Ref. 13). The quantity of Pin m III in western white pine seedlings was shown to be seasonally regulated; increasing as fall progressed to a maximum in the winter months, and reducing to lowest levels in the summer months (Ekramoddoullah et al. 1995, Ref. 12). Pin m III has also been shown to be associated with frost hardiness of western white pine.

SUMMARY OF THE INVENTION

In order to investigate the potential anti-freeze property of Pin m III, the present inventors undertook the molecular cloning and characterization of the gene encoding Pin m III, which is one of the objects of this invention. An expression cDNA library from poly(A)+mRNA of pine needles was generated. The gene encoding Pin m III was cloned by antibody screening. BLAST search and subsequent sequence comparisons indicated Pin m III is a member of intracellular pathogenesis-related protein. This disclosure describes the complete sequence of the cDNA encoding Pin m III from western white pine, its phylogenetic relationship to other pathogenesis-related proteins, and analysis of its temporal and spatial expression. Furthermore, data are provided to show that Pin m III is also induced by the white pine blister rust fungus.

Accordingly, another object of the invention is to provide methods for determining frost hardiness and blister rust fungus susceptibility in a conifer by detecting the amount of the protein Pin m III in the conifer.

Thus according to one aspect, the invention provides a nucleic acid sequence encoding the protein Pin m III having the amino acid sequence of SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of protein sequence analysis of the deduced amino acid sequence of Pin m III.

FIG. 2 shows the phylogenetic tree of thirty representative members from the intracellular PR protein family. The consensus phylogenetic tree was constructed using the PHYLIP package (Felsenstein, 1989, Ref. 14) based on multiple sequence alignment using CLUSTAL W 1.6 (Thompson et al, 1994, Ref. 43). The numbers at branches are bootstrap values out of 100 (the number of times the group consisting of the species occurred among the trees, out of 100 trees). The following sequences were used to construct the phylogenetic tree: BV1A_BETVE (major pollen allergen BET V I-A from white birch, Betula verrucosa, SWISS-PROT: P15494), BV1F_(—) BETVE (major pollen allergen BET V I-F/I, SWISS-PROT: P43179), BV1M_(—) BETVE (major pollen allergen BET V I-M/N, SWISS-PROT: P43186), BV1B_(—) BETVE (major pollen allergen BET V I-B, SWISS-PROT: P45431), BV1C_(—) BETVE (major pollen allergen BET V I-C, SWISS-PROT: P43176), BV1D_(—) BETVE (major pollen allergen BET VI-D/H, SWISS-PROT: P43177), BV1E_(—) BETVE (major pollen allergen BET V I-E, SWISS-PROT: P43178), BV1G_(—) BETVE (major pollen allergen BET V I-G, SWISS-PROT: P43180), BV1J_(—) BETVE (major pollen allergen BET V I-J, SWISS-PROT: P43183), BV1K_(—) BETVE (major pollen allergen BET V I-K, SWISS PROT: P43184), BV1L_(—) BETVE (major pollen allergen BET V I-L, SWISS-PROT: P43185), DRR1_PEA (disease resistance response protein PI176 from garden pea, Pisum sativum, SWISS PROT: P13239), DRR3_PEA (disease resistance response protein PI49, SWISS-PROT: P14710), DRR4_PEA (disease resistance response protein DRRG49-C, SWISS-PROT: P27047), MAL1_(—) MALDO (major allergen MAL D I from apple, Malus domestica, SWISS-PROT: P43211), MPA1_(—) CARBE n(major pollen allergen CAR B I, isoforms 1A and 1B from hornbeam, Carpinus betulus, SWISS-PROT: P38949), MPA2_(—) CARBE (major pollen allergen CAR B I, isoform 2, SWISS-PROT: P38950), MPAA_(—) CORAV (major pollen allergen COR A I, ISOFORMS 5, 6, 11 and 16 from European hazel, Corylus avellana, SWISS-PROT: Q08407), MPAG_(—) ALNGL (major pollen allergen ALN G I alder, Alnus glutinosa, SWISS-PROT: P38948), MPAG_(—) APIGR (major allergen API G I (API G 1) from celery, Apium graveolens, SWISS-PROT: P49372), PR11_(—) PETCR (pathogenesis-related protein A (PR1-1) from parsley, Petroselinum crispum, SWISS-PROT: P19417), PR13_(—) PETCR (pathogenesis-related protein B (PR1-3), SWISS-PROT: P19418), PR2_(—) PETCR (pathogenesis-related protein 2, SWISS-PROT: P27538), PR1_(—) ASPOF (pathogenesis-related protein 1 (AOPR1) from garden asparagus, Asparagus officinalis, SWISS PROT: Q05736), PR1_(—) PHAVU (pathogenesis-related protein 1 (PVPR1) from bean, Phaseolus vulgaris, SWISS-PROT: P25985), PR2_(—) PHAVU (pathogenesis-related protein 2 (PVPR2), SWISS-PROT: P25986), PRS1_(—) SOLTU (pathogenesis-related protein STH-21 from potato, Solanum tuberosum, SWISS-PROT: P17641), PRS2_(—) SOLTU (pathogenesis-related protein STH 2, SWISS-PROT: P17642), SAM2_SOYBN (stress-induced protein SAM22 from soybean, Glycine max, SWISS-PROT: P26987).

FIG. 3 shows the results of Southern blot analysis of genomic DNA of western white pine and related species. Thirty μg of genomic DNA was used in each overnight digestion. Lanes 1-4: genomic DNA of western white pine digested with Xba I (lane 1), Bam HI (lane 2); Hind III (lanes 3 and 8), Eco RI (lane 4); genomic DNA of eastern white pine digested with Hind III (lane 5); ponderosa pine genomic DNA digested with Hind III (lane 6); and tobacco genomic DNA digested with Hind III (lane 7),

FIG. 4 shows RNA gel blot analysis of seasonal expression of Pin m III transcripts in the foliage of one year old western white pine seedlings. Total RNA was isolated, electrophoresed, and transferred to membranes as described hereinafter in “Materials and Methods”. Pin m III transcript of ca. 0.9 kb was detected using a ³²P radiolabeled probe of the full length Pin m III insert. The membranes were exposed for one week. Samples were collected from seed lot 2888. Lane 1, August, 1992; lane 2, September 1992; lane 3, October 1992; lane 4, November 1992; lane 5, December 1992; lane 6, January 193; lane 7, February 1993; lane 8, March 1993; lane 9, April 1993; lane 10, May 1993; lane 11, June 1993; lane 12, July 1993.

FIG. 5 shows Western immunoblot analysis of seedlot 2881 foliage proteins showing the seasonal variation of the Pin m III content. Five μg of total protein was loaded in each lane. The blot was probed with anti-Pin l I antibody. Lane 1, June 1993 foliage sample; lane 2, May, 1993; lane 3, April 1993; lane 4, March 1993 foliage sample; lane 5, February 1993; lane 6, January 1993; lane 7, December 1992; lane 8, November 1992; lane 9, October 1992; lane 10, MW marker; lane 11, September 1992; lane 12, August 1992 and lane 13, July 1992.

FIG. 6 shows RNA gel blot analysis of Pin m III transcript levels in white pine seedling tissues. Samples were collected from healthy western white pine seedling (seedlot 3144) in September 1996. Lane 1, current year foliage; lane 2, one year old foliage; lane 3 current year twig; lane 4, one year old twig and lane 5, root system from the seedling.

FIG. 7 shows RNA gel blot analysis of Pin m III homologue (i.e. Pin l I transcript levels in white pine blister fungus infected sugar pine foliage. Lane 1, infected spots of tree R4 (resistant); lane 2, health foliage of tree R4 (resistant) needles; lane 3, infected spots of tree S4 (susceptible) and lane 4, healthy foliage of tree S4 (susceptible).

FIG. 8 shows Western immunoblot analysis of Pin m III level (lanes 1-5) in different tissues of western white pine from seedlot 3144 collected in September 1996 and Pin l I level (lanes 6-11) in sugar pine. Lane 1, current year foliage; lane 2, one year old foliage; lane 3, current year twig; lane 4, one year old twig; lane 5, roots; lane 6, infected spots of tree S4 (susceptible); lane 7, healthy portion of infected foliage of tree S4; lane 8, healthy foliage of tree S4; lane 9, infected spots of tree R4 (resistant); lane 10, healthy portion of infected foliage of seedling R4 and lane 11, health foliage of tree S4.

FIG. 9 shows the level of Pin m III in the bark samples collected on different dates from mature resistant and susceptible western white pine treees. Error bars represent the standard error of mean quantity of Pin m III in a given number (n) of trees. In case of susceptible trees bark samples were taken from healthy areas away from the cankered tissues. Proteins were separated (5 μg/lane) by SDS-PAGE followed by Western blot. Pin m III was quantified by probing the blot with rabbit anti-Pin l I antibody and by scanning the immunoblot.

DETAILED DESCRIPTION OF THE INVENTION MATERIAL AND METHODS

Mature Western White Pine

Bark samples (all tissues outside the xylem, including epidermis, periderm, and phloem) were collected from both resistant and susceptible clones grown at Plantation 4/Plot B [Hunt et al. 1987, Ref. 19) located at Lens Creek, Vancouver Island, British Columbia. Selection for resistance (clones G-8, G161 and B643) was based upon so-called ‘mature resistance’. Such clones were difficult to infect in a diseased garden; i.e., growing with C. ribicola infected Ribes ssp (Porter, 1960, Ref. 34), and ramets have maintained high level of field resistance displaying phenotypic resistance (Hunt and Meagher 1989, Ref. 21). Sampled ramets lack cankers. Similarly tested susceptible clones (G27 and 6149) were also sampled and sampled ramets had four or more cankers. Ramets were 31-33 years-old in 1991. Along with the clonal samples three ‘natural’ trees were sampled as follows: “resistant” NO-7 (no canker and 7-year old), and susceptible N6-23 (6 cankers and 23 year-old) and N2-15 (2 cankers, 15 year old). All samples were taken from healthy trunk bark in April 1991, October 1993, February 1994 and May 1994.

Western White Pine Seedlings

Two-year old white pine seedlings, were inoculated in August 1992, by suspending Ribes leaves over seedlings (Hunt, 1988, Ref. 20) with a composite inoculum from six coastal British Columbia sources of the blister rust fungus. The seedlings were grown under natural day length and temperature conditions outdoors at Lake Cowichan Research Station (Vancouver Island). The following winter, the seedlings were transferred from styrofoam blocks to either one gallon pots (resistant) or to beds (susceptible). Both susceptible (20 cankered individuals) and resistant (23 individuals displaying slow canker growth; see Hunt 1997, Ref. 22) seedlings were from eight families undergoing screening for blister rust resistance for coastal British Columbia (Meagher et al.1995, Ref. 29). Samples of bark, twig and foliage of these seedlings were collected in July 1995.

Protein Extraction

Proteins were extracted as described by Ekramoddoullah (1991, 1993, Refs. 8 and 9) with minor modifications. Samples of foliage, bark and twig were lyophilized and ground to powder in liquid nitrogen with a mortar and pestle, after which 50 mg of powder was extracted with 0.7 mL of extraction solution (ES) (4% SDS, 5% sucrose, 5% mercaptoethanol) for 10 min at room temperature with gentle stirring. The extract was centrifuged at 10,000 g for 15 min, and the clear supernatant was heated to 100° C. for 3 min and then cooled to room temperature. Proteins were precipitated by adding cold (−20° C.) acetone (8× volume of the supernatant); precipitation was allowed to continue for 1 h, (at −20° C.) after which the sample was centrifuged at 10,000 g. The pellet was resuspended in 0.2 mL of ES, centrifuged at 10,000 g for 15 min, and the protein content of the supernant was determined (Ekramoddoullah and Davidson 1995, Ref. 11) using bovine serum albumin as a standard. Briefly, the protein solution and standard were spotted on a polyvinylidene difluoride membrane IMMOBILON-PT™, available from Millipore Canada Ltd., Toronto, Canada. The membrane was stained with 0.1% Coomassie blue R-250 (Bio-Rad Laboratories, Richmond, Calif., USA) in 50% methanol for 8 min, and then destained in 50% methanol:10% acetic acid for 8 min at room temperature. The membrane was then rinsed with water for 10 min and scanned using a laser scanner (Molecular Dynamics, model 110A, Sunnyvale, Calif., USA) interfaced with a workstation (SPARK 1™, Sun Microsystems of Canada Inc. Vancouver, B.C., Canada) and PDI (Protein+DNA imageWare systems, Huntington Station, N.Y., USA) for membrane blot processing with the software program ONED™. Scanning, detection and quantification were performed according to the PDI instruction manual.

Quantification of Pin m III by Western Immunoblot

SDS-PAGE was carried out in a protein slab cell apparatus (Bio-Rad) utilizing 0.75-mm thick 12% gel and the Laemmli buffer system (Laemmli 1970, Ref. 23). A sample volume of 25 μL (containing 5-20 μg protein) was applied in each well. Proteins separated by SDS-PAGE were electrophoretically transferred (Towbin et al. 1979, Ref. 44) from the gel onto IMMOBILON-P membrane. Following transfer of the separated proteins, the membrane was probed with antibody. The scanning of immunoblots and the preparation of the antibody was described elsewhere (Ekramoddoullah et al. 1995, Ref. 12). The antibody was prepared against a synthetic peptide of 17 amino acid residue of a sugar pine protein Pin l I, but reacts to Pin m III. A sample containing sufficient amount of Pin m III (as determined previously by Western immunoblot) was used as a reference standard to quantify this protein in all samples involving several Western immunoblots. The levels of Pin m III was normalized with respect to this reference standard and expressed as optical density (OD) unit.

Statistical Analysis

Data were analyzed by Student-t and Kruskal-Wallis tests using SAS program (SAS Institute Inc. 1989, Ref. 36).

Plant Material Collection and Treatment for RNA Preparation

The western white pine needles from one year old seedlings were collected in the winter of 1994 and stored at −80° C. The foliage samples of seedlots 2881, 2888 and 3159 for total RNA extractions were the same samples used in the protein analysis as previously described (Ekramoddoullah et al. 1995, Ref. 12). Needle samples used for the expression cDNA library were collected in January, 1996 from a mature western white pine tree growing at the campus of University of Victoria (Victoria, BC, Canada). To analyze tissue specific expression samples from one year old western white pine seedlings (seedlot 3144) were collected in September, 1996 from the shelter house at the Pacific Forestry Centre (Victoria, BC, Canada), frozen in liquid nitrogen and then lyophilized. Current year needles and one year old needles, their corresponding twigs and the roots from the seedling were collected in September 1996 and used in the extraction of total RNA and protein. Sugar pine (Pinus lambertiana) needles (pooled healthy needles alone; pooled infected spots, i.e.+lesions; pooled remainings of infected needles without lesions) from resistant tree (R4) or susceptible tree, (S4) were collected in September 1996 from the shelter house at the Pacific Forestry Centre and treated the same as the one-year-old western white pine seedling. Sugar pine trees were seven years old which were inoculated with the blister fungus in September, 1995.

RNA Extraction

For the cDNA library construction, total RNA was extracted from 10 g western white pine needles collected in January 1996 from the campus of the University of Victoria by the method of Schultz et al. (1994, Ref. 39). The needles were ground to a fine powder in liquid nitrogen with a mortar and pestle. The powder was then transferred to cooled centrifugation tubes, extraction buffer added, and further homogenized in the polytron. The RNA was extracted as described (Schultz et al 1994, Ref. 39). Poly(A)+RNA was isolated by the mRNA Purification Kit from Pharmacia Biotech. For northern blot analysis, total RNA was extracted based on the method of Wang and Vodkin (1994, Ref. 50) with modification. The mRNA concentration was determined spectrophotometrically and also by agarose gel electrophoresis.

Construction of Expression cDNA Library and Antibody Screening of the Library

An expression cDNA library was constructed in γt22A from poly(A)+RNA of western white pine needles according to the manufacture's protocol (GibcoBRL, Gaithersburg, Md., U.S.A.). The library was screened with anti -Pin l I antibody. Three initial positive plaques were identified and one was purified through three rounds of screening. The cDNA insert was subcloned into pBluescript KS+ (Stratagene, La Jolla, Calif., U.S.A.).

Northern Blot Analysis

In northern blot analysis, 20 μg total RNA per lane was electrophoretically separated on 1.2% agarose gels containing 2.2M formaldehyde (Sambrook et al., 1989, Ref. 38), photographed, and transferred to Zeta-Probe GT membranes according to the manufacturers instructions (BIO-Rad, Mississauga, Ontario, Canada). The Pin m III cDNA insert and the genomic DNA 18S ribosomal insert were labeled by random priming with [α-³²P]dCTP and hybridized according to the maunufacturer (Gibco-BRL). The membranes were washed under high stringency also according to the manufacturer (BIO-RAD). Membranes were exposed to BioMax film (Kodak, Rochester, N.Y.) for 3.5 hours for 18S rRNA gene probe, for 3-7 days for Pin m III probe with intensifying screens at −80° C. Northern blot analysis was repeated three times for the seasonal expression analysis and repeated twice for the analysis of tissue specificity and for the infected foliage.

Genomic DNA Extraction and Southern Analysis

Genomic DNA was extracted by the CTAB method. 30 μg aliquots of DNA was digested with restriction enzymes overnight at 37° C., electrophoresed through 0.8% agarose gels and blotted onto Zeta-Probe GT membranes. The Pin m III cDNA insert (EcoR I fragment) was labeled, hybridized and washed as in Northern blot analysis. Southern analysis was repeated three times.

DNA Sequencing

The Lambda DNA was purified by the QIAGEN Lambda Maxi Kit (QIAGEN GmbH, Hilden, Germany). The inserts from the γgt22A vector was purified by fractionating the digestion mixture on 1% agrose gel. The insert was then purified by QIAquick Gel Extraction Kit (QIAGEN GmbH, Hilden, Germany). The cDNA insert was subcloned into pBluescript KS+ vector (Stratagene, La Jolla, Calif.). Inserts were sequenced on both strands by the dideoxy method (Sequenase Version 2.0, US Biochemical, Cleveland, Ohio, USA) with T3 and T7 primers and other internal primers based on derived sequences and synthesized using the Applied Biosystems PCR-Mate DNA Synthesizer (Applied Biosystems, Inc., Foster City, Calif., U.S.A.).

Immunoscreening of cDNA Library

Anti -Pin l I antibody (Ekramoddoullah et al. 1995, Ref. 12) was used in the immunoscreening of the expression cDNA library. Immunoscreening was conducted following the Stratagene's picoBlueTM immunoscreening instruction manual with some modification. Blocking agents TTBS, gelatin, skim milk and BSA were tested for the antibody screening. Best results were obtained using 5% skim milk powder (5% w/v in TBS) overnight. Western immunoblot analysis was done as described above.

Results

Construction of cDNA Library

Total RNA purified by the method of Wang & Vodkin (1994, Ref. 50) was pooled and subjected to oligo-dT column purification for poly(A)+mRNA. The purified poly(A)+mRNA had 260/280 OD ratio of 1.40 to 1.63. Subsequently, these poly(A)+mRNA were used for the cDNA synthesis and cDNA library construction of five libraries according to the instruction manual of Stratagene. Generated libraries were low in total independent plaques which indicated that RNA was perhaps contaminated and inhibited the efficiency of the cDNA library construction. Finally, the protocol described by Schultz et al. (1994, Ref. 39) was adopted for extraction of total RNA from winter needles. This method gave a lower yield but much cleaner total RNA with 260/280 OD ratio of 1.88 to 2.0. The purified poly(A)+mRNA had 260/280 OD ratio of 1.70 to 1.81. UV scanning confirmed the existence of the 260 nm nucleic acid peak. By combining the Gibco-BRL size fractionation column (from Gibco-BRL SuperScript lambda system) with the Stratagene cDNA synthesizing kit (Stratagene UniZAP XR system), we made an excellent cDNA library using Stratagene's expression vector UniZAP XR. A total of 1×10⁶ independent plaque forming units (pfu) were obtained in the cDNA library.

Cloning and Characterization of Pin m III cDNA

Immunoscreening identified three positive clones in the first round. The second and third round immunoscreening of one of the three clones confirmed a specific signal. After three rounds of consecutive screening of the gt22A western white pine needle cDNA expression library with anti-Pin l I antibody, the sequence of a cDNA encoding Pin m III was obtained. The cDNA Pin m III sequence (SEQ ID No: 1) contained 806 bp, a 5′ untranslated region of 54-bp followed by an open reading frame of 486-bp Pin m III and followed by a 286-bp 3′ untranslated region including the poly(A) tail. The deduced open reading frame codes for 161 amino acids. The predicted molecular weight of Pin m III is 18002.30 Da. In the 3′ noncoding region, a putative polyadenylation signal (AATAAA) was located at position 590. The poly(A) tail was located 221 nucleotides downstream from the TAG translation termination codon. The deduced amino acid sequence of the Pin m III had an overall excess of two strongly acidic (−) residues (D,E) in relation to strongly basic (+) amino acids (K,R) (22 acidic versus 20 basic amino acids). It had 50 hydrophobic amino acids (A,I,L,F,W,V) and 44 polar amino acids (N,C,Q,S,T,Y). The predicted isoelectric Point is 5.534. At pH 7.0 the charge is −2.024 . The complete amino acid sequence of Pin m III was derived on the basis of the nucleotide sequence of a cDNA clone obtained from the western white pine winter needle mRNA and confirmed by the partial N-terminal protein sequence of Pin m III. Protein sequence analysis revealed the presence of α, β, and coil structures (FIG. 1). More importantly, Pin m III is very hydrophilic (FIG. 1)—a desirable characteristic of anti-freeze proteins.

Pin m III is Similar to Pathogenesis-Related (PR) Proteins

The BLAST search (Altschul et al., 1990, Ref. 1) of the nucleotide and protein databases revealed similarities between the sequences of Pin m III and a group of intracellular pathogenesis-related (PR) proteins and a group of major tree pollen allergens (FIG. 2). Pin m III shares from 22 to 36.7 percent similarity (according to DNASTAR, using Clustal method with PAM250 residue weight table) with the representative PR and allergen family members from angiosperm plants. The highest similarity is with the PR1 from asparagus (36.7%). The IPRs form a phylogenetic subgroup (except the celery allergen) while the allergens form a separate subgroup (FIG. 2).

A Small Multigene Family Codes for Pin m III in Western White Pine

FIG. 3 shows the Southern analysis of genomic DNA of western white pine, eastern white pine and ponderosa pine (Pinus ponderosa Dougl.) that were digested with four different restriction enzymes: Xba I, Bam HI, Hind III, Eco RI. For western white pine, the Xba I digestion gives at least seven different bands, while Eco RI and Hind III digestion showed at least four bands. Bam HI digestion also gives multiple bands, at least four distinct bands (on another repeated blot). These results would indicate that Pin m III in western white pine is encoded by a small polymorphic family of 4 to 8 genes and the western white pine genome has a small gene family encoding Pin m III. Eastern white pine (Pinus strobus L.) genome digested with Hind III or Eco RI (data not shown) gave a similar banding pattern as that of western white pine. On the other hand, genomic DNA of ponderosa pine had multiple bands differing in sizes as compared to that of western white pine. Tobacco (Nicotiana tabacum L.) genomic DNA did not cross-hybridize with Pin m III probe (lane 7, FIG. 3) suggesting the similarity in PR gene family between conifers and dicotyledonous plants is low.

Seasonal Regulation of the Pin m III Transcript

Northern analysis showed that the highest message was in October (FIG. 4) while Western immunoblot analysis showed that the highest amount of the Pin m III is in January (FIG. 5). The trend of the mRNA is the same among three repeated Northern analysis.

Tissue Specific Expression of Pin m III

Pin m III is highly expressed in the root although the amount of protein in the foliage or stem is low (FIGS. 6, 7 and FIG. 8).

Presence of the Pin m III Homologue Transcripts in the Infected Sugar Pine Needles Western and Northern analysis of blister rust infected resistant and susceptible sugar pine needles showed that both the level of Pin m III protein and mRNA are high in the infected needles than the healthy needles (FIGS. 7 and 8). These data would suggest that Pin m III is induced by the fungal infection.

Identification of Pin m III as Pathogenesis-Related Protein

Mature Western White Pine

The quantity of Pin m III in the bark samples that were collected in April 1991 from four resistant and four susceptible white pine trees is shown in FIG. 9. The level of Pin m III in susceptible trees was significantly (p=0.0209 by Kruskal-Wallis test) higher than that of resistant trees. A similar siginificant difference (p=0.0339 by Kruskal-Wallis test )was obtained when these trees were sampled in October 1993 and in February 1994 samples. Eleven ramets representing 4 clones of resistant trees along with three susceptible trees were sampled in May 1994. Again, the level of Pin m III in these resistant clone was significantly (p=0.0102 by Kruskal-Wallis test) low as compared to susceptible trees.

Western White Pine Seedlings

Cankered bark tissues of all 20 susceptible western white pine seedlings had a significant (p=05 by Student-t test) level of Pin m III ranging from 0.248 to 2.749 O.D. (see Table I below). This protein was also detected at a much lower level in healthy tissues or tissues outside the canker margin in all these trees. When the marginal and healthy tissues were analyzed by increasing the sample load from 5 to 20 μg, the level of Pin m III was slightly higher with an average of 0.222 for marginal and 0.163 for healthy tissues.

TABLE I Level of Pin m III (OD) in bark samples of main stem of susceptible western white pine seedlings. Proteins were separated (5 mg/lane) by SDS- PAGE followed by Western blot. Pin m III was quantified by probing the blot with rabbit anti-Pin l I antibody and by scanning the immunoblot Seedling Outside margin number* Cankered tissue of cankered tissue Healthy tissue 3115B 19-3 1.626** 0.052 0.046 3115B 87-1 2.715 0.108 0.495 3110 37-1 1.96 0.032 0.467 3110B 24-2 1.884 0.04 0.029 3104B 69-2 1.604 0.023 0.026 3099 56-4 1.8 0.391 0.246 3111B 16-3 1.32 0.028 0.027 3111B 34-5 0.937 0.027 0.017 3099 19-1 1.511 0.064 0.025 3115B 19-4 1.026 0.102 0.082 3104B 51-3 1.352 0.354 0.122 3115B 87-3 1.998 0.124 0.022 3104B 69-5 1.148 0.031 0.03 3111B 21-4 0.491 0.02 0.024 3115B 37-1 1.616 0.024 0.034 3099 49-4 1.019 0.07 0.058 3104B 69-1 0.829 0.693 0.174 3115B 19-1 0.248 0.012 0.019 3093 16-4 0.572 0.021 0.025 3110B 129-4 2.749 0.672 0.121 Mean*** 1.4203^(A) 0.1444^(B) 0.1044^(B) *first four digits followed by letter or space refer to the seed source (i.e. family) of the seedlings while last three or four digits identifies the individual seedling. **O.D. units per mm ***means with same letter are not significantly different

The level of Pin m III in twigs and foliage of these seedlings is shown in Table II.

TABLE II Level of Pin m III (OD) in twig and foliar samples of susceptible western white pine seedlings. Proteins were separated (20 μg/lane) by SDS-PAGE followed by Western blot. Pin m III was quantified by probing the blot with rabbit anti-Pin l I antibody and by scanning the immunoblot foliage foliage Current foliage of of 1- of 2- Seedling year 1-year 2-year Current year old year old number* twig old twig old twig year twig twig twig 3115B19-3 0** 0 2.389 0 0 0 3115B87-1 0.058 0.675 3.369 0 0.036 0.023 311037-1 0 0 0 0 0.025 0.028 3110B24-2 0 0 1.545 0 0.019 0.033 3104B69-2 0 0 3.63 0 0 0 309956-4 0 0 2.821 0 0 0 3111B16-3 0 0 0 0 0 0 3111B34-5 0 0 0.164 0 0 0.093 309919-1 0 0 0.055 0 0 0 3115B19-4 0 0 0.802 0 0 0 3104B51-3 0 0 1.5 0 0.096 0.07 3115B87-3 0 0 0 0 0 0 3104B69-5 0 0 0.47 0 0 0 3111B21-4 0 0 0 0 0 0 3115B37-1 0 0 0.049 0 0 0 309949-4 0 0 0 0 0 0 3104B69-1 0 0 0.437 0 0 0 3115B19-1 0 0.042 0 0 0 0 309316-4 0 0 0 0 0 0 3110B129-4 0 0.212 0.263 0 0 0 Mean*** 0.0029^(B) 0.0465^(B) 0.8775^(A) 0^(B) 0.0088^(B) 0.0123^(B) *first four digits followed by letter or space refer to the seed source (i.e, family of the seedlings while last three or four digits identifies the individual seedling. **O.D. units per mm ***means with same letter are not significantly different

These twigs or foliage were devoid of cankers or infection spots during the sampling time. The samples were analyzed at 20 μg protein load per lane. In current year twigs, only one tree had detectable level of Pin m III. In 1-year old twig, three trees had detectable level of Pin m III. In 2-year old twigs thirteen trees had detectable level of Pin m III. None of the foliar samples of current year twig had any detectable level of Pin m III. A low level of Pin m III could be detected in the foliage of four 1-year old twigs and five 2-year-old twigs. Comparative anlysis of the mean values showed that 2-year old twigs had significant (p=0.05 by Student-t test) of Pin m III than that of other tissues.

Table III shows the level of Pin m III in twigs and foliage of western white pine seedlings exhibiting a slow canker growth resistant in their stems. The stem bark of these trees were not sampled, because this would have destroyed the trees which were saved for seed orchards propagation. Pin m III was detectable in the current year twig of one tree, and in the 1-year old twig of six trees and in the 2-year old twig of eight trees. None of these trees had any detectable level of Pin m III in the foliage of current year twig. Pin m III was detectable in the foliage of one-year old twig of three trees, and in the foliage of 2-year old twig of eight trees.

TABLE III Level of Pin m III (OD) in twig and foliar samples of resistant (slow canker growth) western white pine seedlings. Proteins were separated (20 μg/lane) by SDS-PAGE followed by Western blot. Pin m III was quantified by probing the blot with rabbit anti-Pin l I antibody and by scanning the immunoblot foliage of cur- foliage foliage Current 1-year 2-year rent of 1- of 2- Seedling year old old year year old year old number* twig twig twig twig twig twig 3110B1-2 0** 0 0 0 0 0 3115B32-4 0 0 0 0 0 0 3110B76-5 0 0 0 0 0 0.026 3110B3-4 0 0 0.075 0 0 0 3104B117-2 0 0.051 0.487 0 0 0.026 309949-3 0 0 0.051 0 0.219 0 31111B120-2 0 0 0.057 0 0 0 3111B4-3 0 0 0 0 0 0 309940-5 0 0 0 0 0.022 0.03 3111B62-2 0 0 0 0 0 0 3111B58-4 0 0 0 0 0 0 3115B13-2 0 0 0 0 0 0 3104B6-1 0 0.053 0 0 0 0.024 3111B54-2 0 0 0.205 0 0 0 311594-4 0.492 0 0 0 0 0 3111B16-5 0 0 0 0 0 0 3104B51-4 0 0 0 0 0.05 0.042 309316-1 0 0 0.345 0 0 0 3110B13-5 0 0.142 0.399 0 0 0.026 3117B31-1 0 0.044 0 0 0 0.238 309326-4 0 0.501 0 0 0 0.146 309341-3 0 0 0.529 0 0 0 3115B94-4 0 0.618 0 0 0 0 Mean*** 0.022^(B) 0.061^(B) 0.0934^(B) 0 0.0127^(B) 0.0240^(B) *first four digits followed by letter or space refer to the seed source (i.e., family) of the seedlings while last three or four digits identifies the individual seedling. **O.D. units per mm *** means with same letter are not significantly different

Discussion

During our investigations of the molecular analysis of the host pathogen interaction of the white pine blister rust pathosystem (Ekramoddoullah and Hunt, 1993, Ref. 10), a sugar pine protein, Pin l I, was detected in increasing amounts in the fall (Ekramoddoullah et al., 1995, Ref. 12). Using anti-Pin l I antibody, its homologue Pin m III was detected in western white pine. Furthermore, the concentration of Pin m III, which reached its maximum in winter months, was significantly correlated with frost hardiness of western white pine foliage. In order to study the potential anti-freeze property of Pin m III, we constructed an expression cDNA library. Sequence analysis revealed similarity between the predicted Pin m III polypeptide and the asparagus AoPR1, the bean PvPR1 and PvPR2 proteins, the potato pSTH2 protein, the pea PI49 protein, the parsley PcPR1-1 protein and a major pollen allergen from birch (Bet v I). These transcripts have been shown to be induced in response to pathogen attack.

The IPRs are subgrouped while the allergens formed the other subgroups suggesting a divergence of functions inherent to the subgroups. Pin m III is most related to the PR1 from asparagus suggesting its closeness to monocotyledons.

Both Pin m III and Bet v 1 belong to a class of intracellular PR proteins, as revealed by sequences in the protein databases. These proteins are known to be strongly up-regulated in plants by pathogens or by treatment of cell cultures with microbial elicitors. These proteins are present in di-cotyledons, monocotyledons, and in conifers from various taxonomically distantly related plant families. In particular, the deduced amino acid sequence of Pin m III showed a striking similarity to the asparagus PR protein. In parsley, in situ RNA hybridization in fungus-infected parsley leaf tissue demonstrated rapid and massive PR mRNA accumulation around infection sites. These findings are considered to indicate a close correlation between PR mRNA accumulation and the disease-resistance response of the plant and the restriction of fungal growth.

Level of Pin m III in the infected white pine trees confirms the PR nature of Pin m III. Thus repeated sampling over 3 years demonstrated that the quantity of Pin m III in the bark of mature trees was high in susceptible trees during summer. Because the content of Pin m III accumulates in winter months and then decreases in summer months (Ekramoddoullah et al., 1995, Ref. 12), this result would suggest that the high level of Pin m III observed in late spring samples was due to continued presence of the blister rust fungus.

To corroborate this finding further in a larger number trees, we analyzed the quantity of Pin m III in forty three 7-year-old white pine seedlings which were previously inoculated with blister rust fungus as a part of ongoing screening program. Seedlings were selected based on slow canker growth (a form of resistance) vs normal canker growth in their stems. Seedlings from the same families were represented in both groups. Because the difference in the quantity of Pin m III between resistant and susceptible mature trees was found to be more in late spring/early summer samples, sampling in these seedlings was done in summer. The results clearly showed that the content of Pin m III in cankered tissues was highest in susceptible trees indicating that host's cell which are in immediate contact with fungus are capable of producing Pin m III. Consistent with this observation it was found that Pin m III could be detected only in spots of infected needles collected in summer months. Since the cankered tissues of slow canker growth trees could not be analyzed without destroying these trees, we decided to analyze the level of Pin m III in twigs and foliage of both susceptible and slow canker growth seedlings. In general, the quantity of Pin m III was higher in 2-year old twigs than foliage of all seedlings tested. However, a greater percentage of susceptible trees had detectable levels of Pin m III than that of slow canker-growth trees in their 2 year-old twigs (cf Tables II & III). This difference is highly significant by Kruskal-Wallis test (p=0.0179).

The findings that Pin m III which normally accumulates in winter months can also be induced by the fungus is similar to some pathogenesis-related proteins that are induced by both biotic and abiotic stress (Lin et al., 1996, Ref. 24). It is possible that there is a common denominator that perceives signals by two different stresses. For example, in overwintering plants which have to survive frost, limiting water potential could be a factor for its survival. It has been suggested that microbial colonization of plants is also restricted by regulating water potential by the host (Pearce, 1996, Ref. 33). Overwintering grasses that have to survive freezing temperature and desiccation are known to be more resistant to fungal diseases (Tronsmo, 1984, 1985, Refs. 45 and 46; Tronsmo et al., 1993, Ref. 47). Based on the genotypic correlation between freezing tolerance and resistance to the snow molds Typhula ishikariensis and Fusanium nivale (Tronsmo et al, 1993, Ref. 47), it was suggested (Hon et al., 1995, Ref. 18) that the same genetic trait(s) may be involved in both disease and freezing tolerance. There is no evidence at this point that Pin m III provides any protection against the fungal infection; however, sequence similarity with known PR proteins e.g. disease resistance response gene in garden pea and since the expression of a large number pathogenesis-related proteins is temporal and spatial particularly in infection sites would suggest a defensive role of Pin m III in white pine blister rust pathosystem.

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2 806 base pairs nucleic acid single linear cDNA AND ITS ENCODED PROTEIN not provided open reading frame of Pin m III 55....537 1 CCC ACG CTC CGA TTG AAG AAA TAT AAG TAT TGT GTA GTT GCG 42 AGA GAG TTG AAA ATG GTG TCA GGG ACT TCA TCA ACG GAA GAG 84 Met Val Ser Gly Thr Ser Ser Thr Glu Glu 5 10 GTG GTT CAA GTG GAG GCA AGG AGG TTG TGG AAC GCC ACA ACG 126 Val Val Gln Val Glu Ala Arg Arg Leu Trp Asn Ala Thr Thr 15 20 AAA GAC AGC CAC AAC TTC TTG CCA AAG GTT TTG CCC GAA GTT 168 Lys Asp Ser His Asn Phe Leu Pro Lys Val Leu Pro Glu Val 25 30 35 TTC ACT TCT GTC ACC TTA CTT CAA GGA GAT GGA GGC GTC GGC 210 Phe Thr Ser Val Thr Leu Leu Gln Gly Asp Gly Gly Val Gly 40 45 50 ACC GTC AAG CAG CTC AAC TTC ACC CCT GGT AAG AAG GAT TTC 252 Thr Val Lys Gln Leu Asn Phe Thr Pro Gly Lys Lys Asp Phe 55 60 65 AGC TTC ATC AAG GAG CGA GTG GAT GAA CTT GAC CAG GAG AAT 294 Ser Phe Ile Lys Glu Arg Val Asp Glu Leu Asp Gln Glu Asn 70 75 80 TTC GTG TAT AAG TAT ACA GCG ATC GAA GGA GGA CCG CTT GGG 336 Phe Val Tyr Lys Tyr Thr Ala Ile Glu Gly Gly Pro Leu Gly 85 90 AAA CAA CTG AGC TCT GCG TGC TTT GAG GTG AAA TTG ATT CCT 378 Lys Gln Leu Ser Ser Ala Cys Phe Glu Val Lys Leu Ile Pro 95 100 105 AGG AAA GAA GGG GGA TGC GTA GCG AGG TGG ACC TGT AAC TAC 420 Arg Lys Glu Gly Gly Cys Val Ala Arg Trp Thr Cys Asn Tyr 110 115 120 GAA ACT CTT CCT GGT GTT CAA CCT GAC GAA GGA AAA CTA AAA 462 Glu Thr Leu Pro Gly Val Gln Pro Asp Glu Gly Lys Leu Lys 125 130 135 GAG ATA AAG GAA GAT AGC TTT GGC ATG TTG AAG AAA GTG GAG 504 Glu Ile Lys Glu Asp Ser Phe Gly Met Leu Lys Lys Val Glu 140 145 150 CAG TAT CTC CTC TCC AAT CCC AAC TTA TAC TGC TAG ATA TGT 546 Gln Tyr Leu Leu Ser Asn Pro Asn Leu Tyr Cys 155 160 TTACCTACGC ATAAATAGTG TAGAGCGCGC GCTCACCGTG CAAAATAAAG 596 GAGAGTCACG ATATGACTTC CTCCCATCGT CATTGTCGTT TATGGGCTGT 646 AGGATGCCTT TGTTATGTGT GCGCCTGCGA TCGTATCTTT ATCGTTGCGT 696 GATTATGTGT AGTTCCGTGA ATTCAAATCA ATGTCAACGT TCGTTCAGTA 746 TTGTGTGTTC AGAGCAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 796 AAAAAAAAAA 806 161 amino acids amino acid single linear not provided 2 Met Val Ser Gly Thr Ser Ser Thr Glu Glu Val Val Gln Val Glu Ala 5 10 15 Arg Arg Leu Trp Asn Ala Thr Thr Lys Asp Ser His Asn Phe Leu Pro 20 25 30 Lys Val Leu Pro Glu Val Phe Thr Ser Val Thr Leu Leu Gln Gly Asp 35 40 45 Gly Gly Val Gly Thr Val Lys Gln Leu Asn Phe Thr Pro Gly Lys Lys 50 55 60 Asp Phe Ser Phe Ile Lys Glu Arg Val Asp Glu Leu Asp Gln Glu Asn 65 70 75 80 Phe Val Tyr Lys Tyr Thr Ala Ile Glu Gly Gly Pro Leu Gly Lys Gln 85 90 95 Leu Ser Ser Ala Cys Phe Glu Val Lys Leu Ile Pro Arg Lys Glu Gly 100 105 110 Gly Cys Val Ala Arg Trp Thr Cys Asn Tyr Glu Thr Leu Pro Gly Val 115 120 125 Gln Pro Asp Glu Gly Lys Leu Lys Glu Ile Lys Glu Asp Ser Phe Gly 130 135 140 Met Leu Lys Lys Val Glu Gln Tyr Leu Leu Ser Asn Pro Asn Leu Tyr 145 150 155 160 Cys 

We claim:
 1. A nucleic acid sequence encoding the protein Pin m III having the amino acid sequence of SEQ ID NO:
 1. 2. The isolated protein Pin m III comprising the open reading frame, from bases 55 to 537 (amino acids 1-161) of SEQ ID NO:
 1. 3. A method of determining the frost hardiness of a conifer seedling comprising detecting the amount of a protein having the open reading frame, from bases 55 to 537 (amino acids 1-161) of SEQ ID NO:
 1. 4. A method of detecting the presence of blister rust fungus in a conifer comprising measuring the amount of a protein having the open reading frame, from bases 55 to 537 (amino acids 1-161) of SEQ ID NO:
 1. 